Introduction

Immunotherapies have shown promise for the prevention and treatment of various diseases (1–3). For effective vaccination against viral and other pathogen infections and for clinical benefits of cancer immunotherapies, optimizing in vivo functions of CD8+ T effector cells is critical (4–7). However, T-cell effector functions are often weakened in individuals with chronic infections and/or malignancies (8–10). Although adoptive T-cell therapy has shown promise for treating viral infection and cancer (11), the requirement of extensive ex vivo manipulation to expand, activate, and potentially increase homing of effector functions of T cells to tumor sites in the hosts limits its application (8, 9). Even when the T cells have been optimally engineered and activated ex vivo, their activity against tumor cells often fails to persist (12, 13). This is in part caused by the hostile tumor immunologic environment that dampens the efficacies of T cells activated ex vivo (5, 14, 15). It is therefore highly desirable to be able to efficiently upregulate effector functions of CD8+ T cells in vivo not only to reduce the requirement of extensive ex vivo manipulation of T cells but also to circumvent the immunosuppression associated with chronic infections and/or cancer.

We and others have recently identified signal transducer and activator of transcription 3 (Stat3) as negative regulator of Th1 immunity (16–19). In the setting of malignancy, Stat3 is persistently activated not only in tumor cells but also in tumor-associated myeloid cells as well as regulatory T cells (16, 20). Inhibiting Stat3 in either tumor cells or tumor myeloid cells can elicit Th1 antitumor innate and adaptive immune responses, which is accompanied by an increase in tumor-infiltrating CD8+ T cells and decrease in tumor-regulatory T cells (17). However, for potential clinical translation of these findings, it is critical to determine whether targeting Stat3 in myeloid cells can alter the effector functions of adoptively transferred CD8+ T cells.

It has also been shown that certain Toll-like receptor (TLR) signaling activates Stat3, which in turn constrains the magnitude of innate immune responses (21–23). Ablating Stat3 in the myeloid compartment and B cells drastically improves the efficacy of TLR9 agonist CpG-induced antitumor immune responses (24). By conjugating CpG with small interfering RNA (siRNA), we have recently developed a novel in vivo siRNA delivery technology platform achieving targeted delivery and gene silencing in myeloid cells and B cells, as well as immune activation (25). In the current study, we explore the feasibility of using CpG-Stat3siRNA to improve the effector functions of adoptively transferred CD8+ T cells in vivo, thereby developing an approach to alleviate the extensive ex vivo manipulations while improving the antitumor efficacies of transferred T cells.

Mice

Stat3flox mice were kindly provided by S. Akira (Osaka University, Osaka, Japan). Ova TCR (OT-I), Rag1(ko)Momj/B6.129S7, and Mx1-Cre transgenic mice were purchased from The Jackson Laboratory. Stat3flox and Mx1-Cre mice were crossed and treated with poly(inosinic-cytidylic acid) to obtain Stat3 conditional knockout in the hematopoietic system as described previously (26). C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). CD11c(YFP)-Tg(BDC2.5)NOD mice were kindly provided by Dr. Chih-Pin Liu (City of Hope, Duarte, CA). Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with established institutional guidance and approved protocols from the Research Animal Care Committees of the City of Hope.

Flow cytometry

Cell suspensions were prepared from lymph nodes and tumor tissues as described previously, followed by staining with different combinations of fluorophore-conjugated antibodies against CD8, CD69, CD4, CD25, FoxP3, IFN-γ, and granzyme B (BD Biosciences). Flow data were acquired using FACSCalibur (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Intravital multiphoton microscopy

Tumor-bearing mice were anesthetized with an isoflurane/oxygen mixture. Fifteen minutes before imaging procedure, mice were given 100 μg dextran-rhodamine (Invitrogen) and 10 μg Annexin V FITC (BioVision) i.v. Extracellular matrix (ECM) emission signals were given by second harmonic generation at λ[excit] = 890 nm (Coherent Chameleon Ultra II Ti:Sa laser). For recording fluorescein and rhodamine emission, λ[excit] = 860 nm was used, and coumarin emission signals were recorded at λ[excit] = 730 nm. Labeling of CD8OT-I cells with CMAC or CFSE CellTracker was performed according to the manufacturer's instructions. Images were acquired using an Ultima Multiphoton Microscopy System (Prairie Technologies) equipped with Prairie View software and non-descanned Hamamatsu PhotoMultiplier Tubes. Images were collected in a 512 × 512, 16-bit, TIFF format. Composite images were created using Image-Pro Plus professional imaging software (Media Cybernetics).

ELISpot assay

Cells (5 × 105) isolated from tumor-draining lymph nodes (TDLN) of tumor-bearing mice as well as from lymph nodes of naïve mice were seeded into a 96-well filtration plate in the presence or absence of 10 μg/mL peptide (TRP2SVYDFFVWL and OVASIINFEKL, AnaSpec; p15EKSPWFTTL generated by the DNA/RNA and Protein Synthesis Core Facility at the City of Hope) for 24 hours at 37°C. Peptide-specific granzyme B and IFN-γ–positive spots were detected according to the manufacturer's instructions (R&D Systems, Diaclone) and manually counted using a binocular microscope.

In vivo CTL killing assay

Splenocytes of syngeneic animals were harvested and split into two populations. Target cell population was pulsed with 2 μg/mL OVASIINFEKL peptide for 2 hours at 37°C followed by CFSEHI (10 μmol/L) fluorescent labeling, whereas the control cell population remained unpulsed but was labeled CFSELO (1 μmol/L). Equal numbers of CFSEHI and CFSELO cells were mixed and adoptively transferred i.v. into tumor-bearing animals. Each animal received 20 × 106 cells. CTL cytotoxic effects were analyzed by flow cytometry (FACSCalibur).

Stat3 ablation in the myeloid compartment improves antigen-specific CTL maturation and tumor infiltration following adoptive transfer of CD8OT-I cells. A, confocal micrographs of TDLNs of mice with Stat3+/+ and Stat3−/− myeloid cells 24 h after adoptive transfer show CD8OT-I cells (blue, fluorescently labeled using CMAC) and perforin 1 expression (green, immunofluorescent staining). Shown are representative images from two independent experiments. Scale bar, 50 μm. B, lack of Stat3 in myeloid cells results in higher production of granzyme B and IFN-γ by adoptively transferred OVA-specific CD8OT-I T cells. ELISpot assay was performed 24 h after adoptive T-cell transfer into mice with Stat3+/+ or Stat3−/− myeloid compartments. Naïve lymphocytes were included as a control. Columns, mean of two independent experiments using four mice per group as analyzed by one-way ANOVA; bars, SE. C, in vivo CTL killing assay using a pool of OVASIINFEKL-pulsed CFSEHI and off-target CFSELO splenocytes was performed 24 h after adoptive transfer of CD8OT-I T cells to Stat3+/+ (white) and Stat3−/− (gray) tumor-bearing mice. The percentages of CFSE-positive CD8OT-I cells in TDLNs were assessed by flow cytometry at times as indicated. Columns, mean of two independent experiments performed in triplicate per each time point; bars, SE. D, tumor infiltration by fluorescently labeled CD8OT-I T cells 24 h after adoptive transfer into mice with Stat3+/+ or Stat3−/− myeloid compartment was analyzed by flow cytometry. Shown are representative results from one of two experiments using cells pooled from at least three tumors per group.

Because ablating Stat3 in the hematopoietic system in conjunction with CpG administration improves drastically effector functions of transferred CD8+ T cells, we tested CpG-Stat3siRNA as a therapeutic molecule in this setting. We monitored the cellular uptake of red fluorescently labeled CpG-Stat3siRNA in naïve transgenic mice with a yellow fluorescent DC population due to expression of yellow fluorescent protein (YFP) under control of the CD11c promoter. Fluorescent CpG-Stat3siRNA or fluorescent CpG without the siRNA moiety was injected s.c., followed by IVMPM analysis of the inguinal lymph nodes 2 hours after injection. Both CpG and CpG-Stat3siRNA were efficiently uptaken by CD11c(YFP)+ cells (Fig. 3A, top), indicating that the siRNA moiety does not affect cellular internalization.

CTL maturation and tumor infiltration of adoptively transferred CD8+ T cells are enhanced by Stat3 silencing in myeloid cells in vivo. A, top row, in vivo uptake of fluorescently labeled CpG (red) or CpG-Stat3siRNA (C/S, red) by CD11c+ (green) DCs. Naïve CD11c-YFP mice were injected s.c. using equimolar amounts of CpG (middle) or CpG-Stat3siRNA (right) or left untreated (left). The oligonucleotide uptake and DC trafficking into lymph nodes were visualized 2 h later by IVMPM. Red and green channels are shown separately next to the overlay. Scale bar, 50 μm. Bottom row, B16-OVA tumor-bearing mice received adoptive transfer of CD8OT-I T cells in combination with three peritumoral injections of either CpG-Stat3siRNA or CpG-luciferase-siRNA (C/L). Mice without treatment or treated only with CpG-luciferase-siRNA or by adoptive T-cell transfer alone were used as controls. CD8+ T-cell activation determined by the level of CD69 expression was measured by FACS 24 h after the last injection. Data are representative of two independent experiments from pooled TDLNs (n = 4). B, Stat3 mRNA expression and the level of activated Stat3 protein in CD11c+ cells isolated from TDLNs (left) or tumors (right), respectively. Mice were treated three times with indicated siRNAs and evaluated by quantitative real-time PCR and flow cytometry. C, granzyme B and IFN-γ production from TDLN lymphocytes of tumor-bearing mice treated as indicated was assessed by ELISpot assay. Columns, mean (n = 4); bars, SE. Statistically significant differences between analyzed groups were determined by one-way ANOVA. ***, P < 0.001; **, P < 0.01; *, P < 0.05. Data are representative of two independent experiments using pooled TDLN cells (n = 3). D, tumor infiltration by adoptively transferred CD8OT-I T cells was analyzed by flow cytometry. Mice were treated three times as indicated above. Data are representative of two independent experiments using cells pooled from four tumors per group.

Because T-cell therapy is often performed in lymphodepleted setting, and to separate the effects of Stat3 inhibition in myeloid cells on host versus transferred T cells, we addressed the antitumor efficacy of CpG-Stat3siRNA administration combined with T-cell transfer in B16 melanoma tumor–bearing Rag1−/− mice. The tumors in Rag1−/− mice repopulated with CD8+ T cells underwent growth regression on CpG-Stat3siRNA treatment, whereas the tumors in the Rag1−/− mice treated with CpG-luciferase-siRNA/CD8+ T cells continued to grow (Fig. 5A). In contrast, the CpG-Stat3siRNA administration alone did not completely inhibit growth of B16 tumors in wild-type mice, indicating that host CD8 T-cell population is insufficient for a desired antitumor response. Furthermore, adoptively transferred CD8 cells in B16 tumor-bearing Rag1−/− mice treated with CpG-Stat3siRNA displayed increased expression of both granzyme B and IFN-γ on restimulation by natural B16 melanoma antigens ex vivo (Fig. 5B). Moreover, a significant B16 tumor regression was observed in Rag1−/− mice receiving CD8 T cells and CpG-Stat3siRNA, but not in the same mice receiving control CpG-scrambled-RNA or CD8 T-cell therapy alone (Fig. 5C). The expression of both granzyme B as well as IFN-γ protein by adoptively transferred CD8 T cells isolated from TDLNs was considerably increased upon CpG-Stat3siRNA administration (Fig. 5D).

Discussion

Current T-cell therapies, most notably adoptive T-cell therapies, require ex vivo activation, expansion, and/or genetic engineering to generate a desired CTL phenotype. This prolonged and extensive ex vivo requirement not only limits T-cell therapy application but also delays patient treatment. Furthermore, the tumor microenvironment poses a serious threat to dampen the effector functions of transferred T cells and constrains their persistence in the hosts (9). It is therefore highly desirable to identify approaches that can reduce or minimize the requirement for ex vivo manipulation of T cells before transfer and, even more importantly, to circumvent the immunosuppressive tumor milieu that interferes the effector functions of transferred T cells. We show here, using genetic approaches, that inhibiting Stat3 in the myeloid compartment and B cells can facilitate the achievement of this goal.

We have recently developed an siRNA delivery technology involving CpG-siRNA conjugate that facilitates siRNA uptake and gene silencing in myeloid cells and B cells. Although CpG-driven immune activation is thought to be mainly mediated by TLR9-expressing DCs, and TLR9 expression is more restricted among human DCs compared with mouse, macrophages and B cells also serve as antigen-presenting cells (38). In addition, both macrophages and B cells are important components of the tumor microenvironment, producing immunosuppressive and angiogenic/metastatic factors (39–42). At the same time, human B cells and plasmacytoid DCs, which play an important role in generating antitumor immune responses, do express TLR9 (38, 43). These findings support further development of human CpG-Stat3siRNA for the potential use in the setting of adoptive T-cell therapy.

Although our current study only tested CpG-Stat3siRNA to improve the effector functions of adoptively transferred T cells in vivo, the genetic studies presented here suggest the possible use of other Stat3 inhibitors. Currently, no direct Stat3 inhibitors are in clinical trials. This is largely due to the fact that Stat3 is a transcription factor that, unlike tyrosine kinases, lacks enzymatic activity and is difficult to drug. The use of siRNA-based therapy, therefore, is an attractive alternative approach to block Stat3 signaling. On the other hand, Stat3 is a point of convergence for many tyrosine kinase signaling pathways, and certain tyrosine kinase inhibitors in the clinic have been shown to inhibit Stat3 in the tumor microenvironment (44). In particular, sunitinib, which has been shown to reduce immunosuppressive Treg cells and myeloid-derived suppressor cells in both patients and mouse tumor models, can inhibit Stat3 activity (45–48). In addition to Sunitinib, other tyrosine kinase inhibitors are also likely to reduce Stat3 activity, thereby enhancing antitumor immune responses. Our findings strongly suggest that Stat3 targeting, by small molecule or siRNA-based strategies, can improve the efficacy and broaden the applicability of adoptive T-cell therapy.

Disclosure of Potential Conflicts of Interest

The authors have no conflicting financial interests.

Acknowledgments

We thank Dr. Piotr Swiderski (City of Hope, Duarte, CA) for CpG and CpGsiRNA synthesis; Dr. Chih-Pin Liu (City of Hope, Duarte, CA) for generously providing us CD11c(YFP)+ mice; Dr. Yong Liu for superb assistance; and the members of Flow Cytometry Core, the Light Microscopy Core, and Animal Facility at City of Hope for their contributions.

Grant Support: NIH grants R01CA122976 and R01CA146092.

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